Pro-angiogenic Factors VEGF Research Articles

ILT4 facilitates angiogenesis in non-small cell lung cancer.

Antiangiogenic therapy targeting VEGF-A has become the standard of first-line therapy for non-small cell lung cancer (NSCLC). However, its clinical response rate is still less than 50%, and most patients eventually develop resistance, even when using combination therapy with chemotherapy. The major cause of resistance is the activation of complex bypass signals that induce angiogenesis and tumor progression. Therefore, exploring novel proangiogenic mechanisms and developing promising targets for combination therapy are crucial for improving the efficacy of antiangiogenic therapy. Immunoglobulin-like transcript (ILT) 4 is a classic immunosuppressive molecule that inhibits myeloid cell activation. Recent studies have shown that tumor cell-derived ILT4 drives tumor progression via the induction of malignant biologies and creation of an immunosuppressive microenvironment. However, whether and how ILT4 participates in NSCLC angiogenesis remain elusive. Herein, we found that enriched ILT4 in NSCLC is positively correlated with high microvessel density, advanced disease, and poor overall survival. Tumor cell-derived ILT4 induced angiogenesis both invitro and invivo and tumor progression and metastasis invivo. Mechanistically, ILT4 was upregulated by its ligand angiopoietin-like protein 2 (ANGPTL2). Their interaction subsequently activated the ERK1/2 signaling pathway to increase the secretion of the proangiogenic factors VEGF-A and MMP-9, which are responsible for NSCLC angiogenesis. Our study explored a novel mechanism for ILT4-induced tumor progression and provided a potential target for antiangiogenic therapy in NSCLC.

Spatial–temporal order–disorder transition in angiogenic NOTCH signaling controls cell fate specification

Angiogenesis is a morphogenic process resulting in the formation of new blood vessels from pre-existing ones, usually in hypoxic micro-environments. The initial steps of angiogenesis depend on robust differentiation of oligopotent endothelial cells into the Tip and Stalk phenotypic cell fates, controlled by NOTCH-dependent cell–cell communication. The dynamics of spatial patterning of this cell fate specification are only partially understood. Here, by combining a controlled experimental angiogenesis model with mathematical and computational analyses, we find that the regular spatial Tip–Stalk cell patterning can undergo an order–disorder transition at a relatively high input level of a pro-angiogenic factor VEGF. The resulting differentiation is robust but temporally unstable for most cells, with only a subset of presumptive Tip cells leading sprout extensions. We further find that sprouts form in a manner maximizing their mutual distance, consistent with a Turing-like model that may depend on local enrichment and depletion of fibronectin. Together, our data suggest that NOTCH signaling mediates a robust way of cell differentiation enabling but not instructing subsequent steps in angiogenic morphogenesis, which may require additional cues and self-organization mechanisms. This analysis can assist in further understanding of cell plasticity underlying angiogenesis and other complex morphogenic processes.

Spatial-temporal order-disorder transition in angiogenic NOTCH signaling controls cell fate specification.

Angiogenesis is a morphogenic process resulting in the formation of new blood vessels from pre-existing ones, usually in hypoxic micro-environments. The initial steps of angiogenesis depend on robust differentiation of oligopotent endothelial cells into the Tip and Stalk phenotypic cell fates, controlled by NOTCH-dependent cell-cell communication. The dynamics of spatial patterning of this cell fate specification are only partially understood. Here, by combining a controlled experimental angiogenesis model with mathematical and computational analyses, we find that the regular spatial Tip-Stalk cell patterning can undergo an order-disorder transition at a relatively high input level of a pro-angiogenic factor VEGF. The resulting differentiation is robust but temporally unstable for most cells, with only a subset of presumptive Tip cells leading sprout extensions. We further find that sprouts form in a manner maximizing their mutual distance, consistent with a Turing-like model that may depend on local enrichment and depletion of fibronectin. Together, our data suggest that NOTCH signaling mediates a robust way of cell differentiation enabling but not instructing subsequent steps in angiogenic morphogenesis, which may require additional cues and self-organization mechanisms. This analysis can assist in further understanding of cell plasticity underlying angiogenesis and other complex morphogenic processes.

Intermittent Topical Bevacizumab for Treatment of Pediatric Corneal Neovascularization after Keratitis

Introduction : Corneal neovascularization imposes pediatric patients to risk of amblyopia. Current treatment of corneal neovascularization consists of topical steroids, nonsteroidal anti-inflammatory medications, immunomodulatory agents, anti-Vascular Endothelial Growth Factor (anti-VEGF) and few surgical procedures. Anti-VEGF shows promising results in treating corneal neovascularization but is difficultto obtain in developing countries such as Indonesia. We report intermittent topical use of Bevacizumab as antiVEGF in a child experiencing corneal neovascularization in the settings of limited antiVEGF availability.
 Case Illustration : A 5-year-old girl presented with corneal neovascularization after keratitis. Topical fluorometholone 4 times a day for 3 weeks was given with no clinical improvement. An intermittent topical Bevacizumab was started, each episode of treatment lasted 4 days. Corneal neovascularization regressed after 1 episode of treatment and thus continued until a total of 2 treatment episodes. She improved clinically on treatment, with some residual corneal scarring. Visual acuity improved from logMar 0.5 to logMar 0.2 following treatment.
 Discussion : Bevacizumab regresses corneal neovascularization by inhibiting proangiogenic factor VEGF which is upregulated in neovascularization. Topical Bevacizumab ability to penetrate cornea was contributed by defective corneal epithelial overlying neovascularization. Long-term use of antiVEGF may hinder VEGF beneficial role on wound recovery and trigger unwanted side effects.
 Conclusion : Intermittent short-term use of topical bevacizumab may be effective to treat corneal neovascularization during acute period in pediatric patient and is safely tolera

Focal Cerebral Ischemia Induces Global Subacute Changes in the Number of Neuroblasts and Neurons and the Angiogenic Factor Density in Mice.

Background and Objectives: Dissecting the complex pathological cascade of an ischemic stroke in preclinical models is highly warranted to understand the course of this disease in humans. Neurogenesis and angiogenesis are integral for post-stroke recovery, yet it is not clear how these processes are altered months after an ischemic stroke. In this study, we investigated the changes that take place subacutely after focal cerebral ischemia in experimental adult male mice. Materials and Methods: Male 12-week-old C57BL/6 mice underwent a 60 min long fMCAo or sham surgery. Two months after the procedure, we examined the immunohistochemistry to assess the changes in neuroblast (DCX) and differentiated neuron (NeuN) numbers, as well as the density of the pro-angiogenic factor VEGF. Results: We found decreased neuroblast numbers in both brain hemispheres of the fMCAo mice: by more than 85% in the dentate gyrus and by more than 70% in the subventricular zone. No neuroblasts were found in the contralateral hemisphere of the fMCAO mice or the sham controls, but a small population was detected in the ipsilateral ischemic core of the fMCAo mice. Intriguingly, the number of differentiated neurons in the ipsilateral ischemic core was lower by 20% compared to the contralateral hemisphere. VEGF expression was diminished in both brain hemispheres of the fMCAo mice. Conclusions: Our current report shows that focal cerebral ischemia induces changes in neuroblast numbers and the pro-angiogenic factor VEGF in both cerebral hemispheres 2 months after an fMCAo in mice. Our data show that focal cerebral ischemia induces a long-term regenerative response in both brain hemispheres.

Targeted Inhibition of Myeloperoxidase (MPO): A New Therapeutic Strategy for the Treatment of Multiple Myeloma

Despite major improvements in therapeutic strategies for patients with multiple myeloma (MM), effective treatment still remains a persistent challenge, as patients ultimately relapse and succumb to the disease. In the last decade, studies have highlighted the reciprocal interaction between MM plasma cells (PC) and the bone marrow (BM) microenvironment in regulating immune evasion, disease progression and persistence. As MM PC rely on BM stromal cells and their secreted factors for their survival and growth, the therapeutic targeting of the BM microenvironment may prove to be a novel and successful strategy for myeloma care in the future. Myeloid-derived suppressor cells (MDSC), a heterogenous population of myeloid cells are described to promote MM progression through immunosuppression and induction of angiogenesis. Myeloperoxidase (MPO), a key inflammatory enzyme important in host defence, is reported to be the most highly upregulated gene in MDSCs in murine cancer models. Recently, the accumulation of MPO within the tumour microenvironment has attracted much attention with a number of studies describing a role for MPO in regulating cancer development due to its potent pro-oxidative and proinflammatory properties. Our most recent findings have revealed new functional roles for myeloid-derived MPO in the BM microenvironment of MM. Specifically, we demonstrate that myeloid cell populations are increased within the BM of 5TGM1 tumour-bearing mice and that MM PC may directly influence Mpo gene expression in BM-derived myeloid cells. Mechanistically, we report that MPO has the capacity to induce the expression of key MM growth factors, and exerts potent immune suppression by inhibiting anti-tumour T-cell responses. Remarkably, in the syngeneic KaLwRij/5TGM1 mouse model of MM, targeted inhibition of MPO with the suicide substrate 4-Aminobenzoic acid hydrazide (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Here, we investigate for the first time the efficacy of an orally bioavailable irreversible small molecule inhibitor of MPO (MPOi) in the preclinical Vk*MYC murine model of myeloma. Twelve-week-old C57BL/6J mice were intravenously inoculated with Vk*MYC (Vk14451-GFP) cells and tumour progression was monitored by serum paraprotein electrophoresis (SPEP), whilst endpoint GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. To characterise myeloid cell populations and associated Mpo expression in the Vk*MYC tumour landscape, we utilised flow cytometry to quantitate CD11b+ cells and used magnetic activated cell separation to isolate these populations and characterise the expression of Mpo by qPCR. Our studies confirm that CD11b+ myeloid cells are significantly increased in the BM of Vk*MYC tumour-bearing mice (p<0.01), accompanied by an upregulation of MPO mRNA expression (p<0.001). To assess the efficacy of MPOi in MM, mice were treated with MPOi or vehicle alone twice daily by oral gavage, with treatment initiated at the time of Vk*MYC inoculation. Notably, mice receiving MPOi presented with significantly reduced endpoint tumour burden (9 weeks post tumour cell inoculation). We observed a 15.2% and 31.4% reduction in MM tumour as determined by hind limb GFP% (p<0.05) and SPEP (p<0.01) respectively, compared to vehicle control. Additionally, mRNA analysis of complete BM revealed an upregulation of the critical cytokine IFN gamma (p<0.05) and the downregulation of the potent proangiogenic factor VEGF (p<0.05) in mice treated with MPOi, suggesting MPO inhibition in MM may be an advantageous means to regulate the BM microenvironment and impede disease progression. However, when MPOi treatment was initiated at first signs of detectable disease, (as identified by the presence of a monoclonal spike by SPEP; 5 weeks post tumour cell inoculation), no difference in endpoint tumour burden was observed. This suggests that targeted inhibition of MPO using MPOi may be more effective in at early stages of MM development. In conclusion, the findings presented in this study indicate that inhibiting MPO activity with MPOi, as a single agent therapy, attenuates MM tumour growth in the Vk*Myc mouse model. With limited therapies used in the clinic that target the stromal microenvironment, these findings suggest MPOi could be investigated as a potential novel treatment option that may be included in combination with current frontline therapeutic agents.

Effect of sunitinib against Echinococcus multilocularis through inhibition of VEGFA-induced angiogenesis

BackgroundAlveolar echinococcosis (AE) is a lethal zoonosis caused by the fox tapeworm Echinococcus multilocularis. The disease is difficult to treat, and an effective therapeutic drug is urgently needed. Echinococcus multilocularis-associated angiogenesis is required by the parasite for growth and metastasis; however, whether antiangiogenic therapy is effective for treating AE is unclear.MethodsThe in vivo efficacy of sunitinib malate (SU11248) was evaluated in mice by secondary infection with E. multilocularis. Enzyme-linked immunosorbent assays (ELISAs) were used to evaluate treatment effects on serum IL-4 and vascular endothelial growth factor A (VEGFA) levels after SU11248 treatment. Gross morphological observations and immunohistochemical staining were used to evaluate the impact of SU11248 on angiogenesis and the expression of pro-angiogenic factors VEGFA and VEGF receptor 2 (VEGFR2) in the metacestode tissues. Furthermore, the anthelmintic effects of SU11248 were tested on E. multilocularis metacestodes in vitro. The effect of SU11248 on the expression of VEGFA, VEGFR2, and phosphorylated VEGFR2 (p-VEGFR2) in liver cells infected with protoscoleces in vitro was detected by western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). The influence of SU11248 on endothelial progenitor cell (EPC) proliferation and migration was determined using CCK8 and transwell assays.ResultsIn vivo, SU11248 treatment markedly reduced neovascular lesion formation and substantially inhibited E. multilocularis metacestode growth in mice. Further, it exhibited high anti-hydatid activity as efficiently as albendazole (ABZ), and the treatment resulted in reduced protoscolex development. In addition, VEGFA, VEGFR2, and p-VEGFR2 expression was significantly decreased in the metacestode tissues after SU11248 treatment. However, no effect of SU11248 on serum IL-4 levels was observed. In vitro, SU11248 exhibited some anthelmintic effects and damaged the cellular structure in the germinal layer of metacestodes at concentrations below those generally considered acceptable for treatment (0.12–0.5 μM). Western blotting, RT-qPCR, and ELISA showed that in co-cultured systems, only p-VEGFR2 levels tended to decrease with increasing SU11248 concentrations. Furthermore, SU11248 was less toxic to Reuber rat hepatoma (RH) cells and metacestodes than to EPCs, and 0.1 μM SU11248 completely inhibited EPC migration to the supernatants of liver cell and protoscolex co-cultures.ConclusionsSU11248 is a potential candidate drug for the treatment of AE, which predominantly inhibits parasite-induced angiogenesis. Host-targeted anti-angiogenesis treatment strategies constitute a new avenue for the treatment of AE.Graphical

Chrysin-Induced Regression of Angiogenesis via an Induction of DNA Damage Response and Oxidative Stress in In Vitro and In Vivo Models of Melanoma.

Despite the progress made in treatments, melanoma is one of the cancers for which its incidence and mortality have increased during recent decades. In the research of new therapeutic strategies, natural polyphenols such as chrysin could be good candidates owing to their capacities to modulate the different fundamental aspects of tumorigenesis and resistance mechanisms, such as oxidative stress and neoangiogenesis. In the present study, we sought to determine whether chrysin could exert antitumoral effects via the modulation of angiogenesis by acting on oxidative stress and associated DNA damage. For the first time, we show a link between chrysin-induced antiproliferative effects, the activation of the DNA damage pathway, and its ability to limit angiogenesis. More specifically, herein, we show that chrysin induces single- and double-stranded DNA breaks via the activation of the DNA damage response pathway: ATM (ataxia-telangiectasia-mutated)/Chk2 (checkpoint kinase 2) and ATR (ataxia telangiectasia and Rad3-related)/Chk1 (checkpoint kinase 1) pathways. Strong activation of this DNA damage response was found to be partly involved in the ability of chrysin to limit angiogenesis and may partly involve a direct interaction between the polyphenol and DNA G-quadruplex structures responsible for the replication fork collapse. Moreover, these events were associated with a marked reduction in melanoma cells' capacity to secrete proangiogenic factor VEGF-A. The disruption of these key protein actors in tumor growth by chrysin was also confirmed in a syngeneic model of B16 melanoma. This last point is of importance to further consider the use of chrysin as a new therapeutic strategy in melanoma treatment.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells

BackgroundBreast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HypothesisEarlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. MethodsCo-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. ResultsData revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. ConclusionIn silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

Cannabidiol Rescues TNF-α-Inhibited Proliferation, Migration, and Osteogenic/Odontogenic Differentiation of Dental Pulp Stem Cells.

Strategies to promote dental pulp stem cells (DPSCs) functions including proliferation, migration, pro-angiogenic effects, and odontogenic/osteogenic differentiation are in urgent need to restore pulpitis-damaged dentin/pulp regeneration and DPSCs-based bone tissue engineering applications. Cannabidiol (CBD), an active component of Cannabis sativa has shown anti-inflammation, chemotactic, anti-microbial, and tissue regenerative potentials. Based on these facts, this study aimed to analyze the effect of CBD on DPSCs proliferation, migration, and osteogenic/odontogenic differentiation in basal and inflammatory conditions. Highly pure DPSCs with characteristics of mesenchymal stem cells (MSCs) were successfully isolated, as indicated by the results of flowcytometry and multi-lineage (osteogenic, adipogenic, and chondrogenic) differentiation potentials. Among the concentration tested (0.1-12.5 µM), CBD (2.5 μM) showed the highest anabolic effect on the proliferation and osteogenic/odontogenic differentiation of DPSCs. Pro-angiogenic growth factor VEGF mRNA expression was robustly higher in CBD-treated DPSCs. CBD also prompted the migration of DPSCs and CBD receptor CB1 and CB2 expression in DPSCs. TNF-α inhibited the viability, migration, and osteogenic/odontogenic differentiation of DPSCs and CBD reversed these effects. CBD alleviated the TNF-α-upregulated expression of pro-inflammatory cytokines TNF-α, interleukin (IL)-1β, and IL-6 in DPSCs. In conclusion, our results indicate the possible application of CBD on DPSCs-based dentin/pulp and bone regeneration.

RNA-binding motif 4 promotes angiogenesis in HCC by selectively activating VEGF-A expression

Increased angiogenesis in the liver plays a critical role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanism underlying increased angiogenesis in HCC is not well understood. Current study was designed to identify the potential angiogenic effect of RNA-binding motif 4 (RBM4)through a small-scale overexpression screening, followed by comparison of the expression level of RBM4 in cancer and adjacent tissues in multiple malignancies to explore the relationship between RBM4 and CD31 protein expression level and related clinical indicators, and understand the role of RBM4 in the hepatocellular carcinoma. To understand the specific mechanism of RBM4 in detail, transcriptome sequencing, mass spectrometry and multiple molecular cytological studies were performed. These cellular level results were verified by experiments in animal models of nude mice. The increased expression of RBM4 in cancer tissues, suggested its use as a prognostic biomarker. The RBM4 expression was found to be strongly correlated with tumor microvessel density. Mechanistically, RBM4 mediated its effects via interaction with HNRNP-M through the latter’s WDR15 domain, which then stabilized RelA/p65 mRNA. Consequently, RBM4 induced the activation of the NF-kB signaling pathway, upregulating the expression of proangiogenic factor VEGF-A. The results confirmed the mechanism by which RBM4 promotes angiogenesis in hepatocellular carcinoma suggesting RBM4 as a crucial promoter of angiogenesis in HCC, helping understand regulation of NF-kB signaling in HCC.

Epithelial membrane protein 2 (EMP2) regulates hypoxia-induced angiogenesis in the adult retinal pigment epithelial cell lines

Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vaso-occlusive diseases. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxic conditions. To study its effects, the RPE cell line ARPE-19 was genetically modified to either overexpress EMP2 or knock down its levels, and RNA sequencing and western blot analysis was performed to confirm the changes in expression at the RNA and protein level, respectively. Protein expression was evaluated under both normoxic conditions or hypoxic stress. Capillary tube formation assays with human umbilical vein endothelial cells (HUVEC) were used to evaluate functional responses. EMP2 expression was found to positively correlate with expression of pro-angiogenic factors HIF1α and VEGF at both mRNA and protein levels under hypoxic conditions. Mechanistically, EMP2 stabilized HIF1α expression through downregulation of von Hippel Lindau protein (pVHL). EMP2 mediated changes in ARPE-19 cells were also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate HUVEC migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potential mediator of angiogenesis in a human RPE cell line. EMP2 levels positively correlate with pro-angiogenic mediators HIF1α and VEGF, and mechanistically, EMP2 regulates HIF1α through downregulation of pVHL. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.

Differential Effects of Resveratrol on HECa10 and ARPE-19 Cells

Age-related macular degeneration (AMD) and diabetic retinopathy are the leading cause of blindness in developed countries. Pathological angiogenesis has a causal role in these eye diseases. Resveratrol (RSV), a plant-derived polyphenol, has anti-proliferative and anti-angiogenic properties that could improve its management. Here, the effects of various concentrations of RSV (1, 5, 10, 50, 100 µM) were compared in two types of cell lines: HECa10 (endothelial cell line) and ARPE-19 (retinal pigment epithelial cell line). We assayed the impact on proliferation rate, viability, cell cycle progression, and secretion of selected proangiogenic factors VEGF and bFGF. We show that lower concentrations of RSV (1, 5, 10 µM) had no effect on proliferation, viability or cell cycle progression in HECa10 cells. However, higher concentrations (50, 100 µM) significantly enhanced the reduction in the cell number and stimulated apoptosis. In ARPE-19 cells, lower concentrations of RSV increased the rate of proliferation, while higher concentrations had no effect on proliferation and viability. Both ARPE-19 and HECa10 cell lines were affected to different degrees in the secretion of proangiogenic cytokines: reducing VEGF and enhancing bFGF secretion. These results suggest that RSV may be useful in the prevention or treatment of pathological angiogenesis in eye disorders.

An In Vitro Engineered Osteochondral Model as Tool to Study Osteoarthritis Environment.

Osteoarthritis (OA) is a joint degenerative pathology characterized by mechanical and inflammatory damages affecting synovium, articular cartilage (AC), and subchondral bone (SB). Several in vitro, in vivo, and ex vivo models are developed to study OA, but to date the identification of specific pharmacological targets seems to be hindered by the lack of models with predictive capabilities. This study reports the development of a biomimetic in vitro model of AC and SB interface. Gellan gum methacrylated and chondroitin sulfate/dopamine hydrogels are used for the AC portion, whereas polylactic acid functionalized with gelatin and nanohydroxyapatite for the SB. The physiological behavior of immortalized stem cells (Y201s) and Y201s differentiated in chondrocytes (Y201-Cs), respectively, for the SB and AC, is demonstrated over 21 days of culture in vitro in healthy and pathological conditions, whilst modeling the onset of cytokines-induced OA. The key metrics are: lower glycosaminoglycans production and increased calcification given by a higher Collagen X content, in the AC deep layer; higher expression of pro-angiogenic factor (vegf) and decreased expression of osteogenic markers (coll1, spp1, runx2) in the SB. This novel approach provides a new tool for studying the development and progression of OA.

Stimulation of human bone marrow mesenchymal stem cells by electromagnetic transduction therapy - EMTT

ABSTRACT Many different pulsed electromagnetic field (PEMF) devises have been clinically used to stimulate healing processes, but many procedures are still without supporting basic research data. The aim of this study was to investigate a new modified pulsed electromagnetic field therapy: electromagnetic transduction therapy (EMTT). EMTT is technically based on high-intensive PEMFs with a magnetic field strength between 80 and 150 mT. The effect of EMTT for a 10-min session three times a week on human bone marrow mesenchymal stem cells (MSCs) was evaluated by assessing cell viability, gene expression of bone regenerative factors and VEGF-A (vascular endothelial growth factor) secretion after 7 and 14 days of treatment. No negative or toxic effects of EMTT on MSCs in vitro were observed in the applied test frame. The VEGF-ELISA at day 7 of EMTT treatment with 80 mT showed a significant higher VEGF concentration compared to untreated control group. In conclusion, high-intensive electromagnetic impulses showed no harmful effects on MSC cultures in our study. The enhancement of the proangiogenic factor VEGF in MSCs on day 7 indicates a substantial role in cell-stimulating effect of EMTT. Further in vitro and in vivo studies should differentiate specific stimulating and regenerating effects of EMTT impulses in soft tissue engineering. Specific electromagnetic characteristics have to be determined to optimize electromagnetic treatment options in orthopedic surgery and traumatology and soft tissue treatment options.

High DNMT1 Expression in Stromal Fibroblasts Promotes Angiogenesis and Unfavorable Outcome in Locally Advanced Breast Cancer Patients

BackgroundActive breast cancer-associated fibroblasts (CAFs) play a leading role in breast carcinogenesis through promoting angiogenesis and resistance to therapy. Consequently, these active stromal cells have significant influence on patient outcome. Therefore, we explored here the role of the DNA methyltransferase 1 (DNMT1) protein in CAF-dependent promotion of angiogenesis as well as the prognostic power of DNMT1 level in both cancer cells and their adjacent CAFs in locally advanced breast cancer patients.MethodsWe applied immunohistochemistry to evaluate the level of DNMT1 in breast cancer tissues and their adjacent normal counterparts. Quantitative RT-PCR and immunoblotting were performed to investigate the role of DNMT1 in regulating the expression of pro-angiogenic genes in active CAFs and also their response to the DNMT1 inhibitors decitabine (DAC) as well as eugenol.ResultsWe have shown that DNMT1 controls the pro-angiogenic potential of CAFs both in vitro and in vivo through positive regulation of the expression/secretion of 2 important pro-angiogenic factors VEGF-A and IL-8 as well as their upstream effectors mTOR and HIF-1α. To confirm this, we have shown that these DNMT1-related pro-angiogenic effects were suppressed by 2 DNMT1 inhibitors decitabine and eugenol. Interestingly, in a cohort of 100 tumors from locally advanced breast cancer patients (LABC), we have shown that high expression of DNMT1 in tumor cells and their adjacent stromal fibroblasts is correlated with poor survival of these patients.ConclusionDNMT1 upregulation in breast stromal fibroblasts promotes angiogenesis via IL-8/VEGF-A upregulation, and correlates well with poor survival of LABC patients.

Increased lung metastasis with sevoflurane in breast cancer surgery is associated with VEGF and elevated vascular permeability by VEGF in a mouse model of human breast cancer.

e13060 Background: An on-going controversy is whether general anesthetics used in cancer surgery substantially influence the outcome of cancer patients. Thousands of cancer patients undergo surgery with hope of cure. However, postoperative metastasis remains a deadly disease that affects millions of lives, even with all the recent therapeutic advancements. Anesthesia is required for surgical resection of solid tumors and we have shown that general anesthetics substantially influences the process of breast cancer lung metastasis through increasing pulmonary permeability in animal models. Our previous study demonstrates that inhaled anesthetic sevoflurane used during resecting primary breast tumors promotes lung metastasis via IL6 pathway in syngeneic model of murine 4T1 breast cancer cells, compared with an intravenous anesthetic propofol. Methods: Xenograft model of human MDA-MB 231 breast cancer of spontaneous metastasis was utilized in this study to evaluate the effect of commonly used general anesthetics including sevoflurane and propofol on breast cancer lung metastasis and explore the potential mechanism. Results: In this study we show that the metastatic promoting effect of sevoflurane is associated with elevated secretion of VEGF and increased vascular permeability in xenograft model of human MDA-MB 231 breast cancer of spontaneous metastasis. Cytokine array profiling reveals that the mice receiving sevoflurane have increased levels of pro-angiogenic factor VEGF in serum and lung tissues. Sevoflurane enhances the expression level of VEGFR on HUVEC cells. Furthermore, sevoflurane disrupts endothelial monolayer in vitro and elevates pulmonary vascular permeability in vivo. The sevoflurane-promoted breast cancer lung metastasis is reversed by DC101, an antibody targeting mouse VEGFR-2. Conclusions: Our results show that the inhaled anesthetic sevoflurane during surgical removal of primary tumor alter the course of metastasis through regulating secretion of VEGF and associated vascular remodeling. The mechanistic study of general anesthetics on cancer cell metastasis establishes the causal link and could provide the potential therapeutic intervention and guide the clinical trials.

Notoginsenoside R1 Promotes Migration, Adhesin, Spreading, and Osteogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stromal Cells.

Cellular activities, such as attachment, spreading, proliferation, migration, and differentiation are indispensable for the success of bone tissue engineering. Mesenchymal stromal cells (MSCs) are the key precursor cells to regenerate bone. Bioactive compounds from natural products had shown bone regenerative potential. Notoginsenoside R1 (NGR1) is a primary bioactive natural compound that regulates various biological activities, including cardiovascular protection, neuro-protection, and anti-cancer effects. However, the effect of NGR1 on migration, adhesion, spreading, and osteogenic differentiation of MSCs required for bone tissue engineering application has not been tested properly. In this study, we aimed to analyze the effect of NGR1 on the cellular activities of MSCs. Since human adipose-derived stromal cells (hASCs) are commonly used MSCs for bone tissue engineering, we used hASCs as a model of MSCs. The optimal concentration of 0.05 μg/mL NGR1 was biocompatible and promoted migration and osteogenic differentiation of hASCs. Pro-angiogenic factor VEGF expression was upregulated in NGR1-treated hASCs. NGR1 enhanced the adhesion and spreading of hASCs on the bio-inert glass surface. NGR1 robustly promoted hASCs adhesion and survival in 3D-printed TCP scaffold both in vitro and in vivo. NGR1 mitigated LPS-induced expression of inflammatory markers IL-1β, IL-6, and TNF-α in hASCs as well as inhibited the RANKL/OPG expression ratio. In conclusion, the biocompatible NGR1 promoted the migration, adhesion, spreading, osteogenic differentiation, and anti-inflammatory properties of hASCs.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Inhibition of FGFR2 enhances chemosensitivity to gemcitabine in cholangiocarcinoma through the AKT/mTOR and EMT signaling pathways

AimTo investigate the oncogenic role of FGFR2 in carcinogenesis in cholangiocarcinoma (CCA) cells. In addition, the feasibility of using FGFR inhibitors in combination with standard chemotherapy was also explored for the chemosensitizing effect in CCA cells. Main methodsFive CCA cell lines were used to screen FGFR2 expression by Western immunoblotting. Two CCA cell lines, KKU-100 and KKU-213A, were knocked down of the FGFR2 gene using siRNA. Cell viability was assessed by the MTS cell proliferation assay. Reproductive cell death was assessed by clonogenic assay. The effects on cell migration and invasion were analyzed by the Transwell chamber method. Cell cycle analysis was performed by flow cytometry. Cell angiogenesis was assessed by HUVEC tube formation and human angiogenesis antibody array analysis. Proteins associated with proliferative and metastatic properties were evaluated by Western blotting. Key findingsKnockdown of FGFR2 suppressed cell growth and colony formation in CCA cells in association with G2/M cell cycle arrest and downregulation of STAT3, cyclin A and cyclin B1. Silencing FGFR2 enhanced the suppressive effect of gemcitabine (Gem) on cell migration and invasion. The combination of infigratinib, an FGFR inhibitor, and Gem, interrupted cell growth, migration, and invasion via downregulation of FGFR/AKT/mTOR pathways and the EMT-associated proteins vimentin and slug. Moreover, the combination also suppressed tube formation together with decreased expression of the proangiogenic factor VEGF. SignificanceInhibition of FGFRs by infigratinib enhanced the antitumor effect of Gem in CCA cells through downregulation of the FGFR/AKT/mTOR, FGFR/STAT3 and EMT signaling pathways.